scholarly journals Establishment of Functional Epithelial Organoid From Human Lacrimal Glands

2020 ◽  
Author(s):  
Sang Yun Jeong ◽  
Woo Hee Choi ◽  
Sookon Lee ◽  
Jong-Moon Park ◽  
Mira Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dry Eye ◽  
Lacrimal Gland ◽  
Culture Media ◽  
Conventional Medicine ◽  
Calcium Influx ◽  
Gene Expression Pattern ◽  
Secretory Function ◽  
Organoid Culture ◽  
Gland Tissue

Abstract Background: Tear deficiency due to dysfunction of the lacrimal gland is one of the major causes of dry eye disease (DED). Artificial tears as conventional medicine often alleviate symptoms, however, they insufficient to prevent the progression of severe DED. Lacrimal gland stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue, but the number of stem cells in the lacrimal gland is pretty low, and 2D long-term cultivation results in the reduction of the differentiation capacity of stem cells. Therefore, 3D lacrimal gland organoids could be an alternative for a DED therapeutic method, which is capable of prolonged growth while maintaining the characteristics of the lacrimal gland tissue.Method: Digested cells from human lacrimal gland tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, expression of specific markers and histological characters were analyzed to prove the lacrimal gland organoids. And then, the secretory function of lacrimal gland organoids was confirmed by calcium influx or proteomics after pilocarpine treatment.Result: Human lacrimal gland organoids were formed and cultured under a modified salivary organoid culture medium, and their histological features and gene expression pattern were similar to normal lacrimal gland tissue. After pilocarpine treatment, their secretory function was confirmed and the secretomes were analyzed by proteomics. More than 70% of the identified proteins were proven to exosome through GO analysis. Additionally, to explore the curability of developed organoids, mouse-derived lacrimal gland organoids were fabricated and transplanted into mouse lacrimal tissue with DED, and we confirmed that organoids were well engrafted in the lacrimal tissue.Conclusion: These results demonstrate that our established lacrimal gland organoids resemble characteristics of normal lacrimal gland tissue and could be used as cell therapy for patients with dry eye syndrome.

scholarly journals Establishment of functional epithelial organoids from human lacrimal glands

2021 ◽  
Author(s):  
Sang Yun Jeong ◽  
Woo Hee Choi ◽  
Seong Gyeong Jeon ◽  
Sookon Lee ◽  
Jong-Moon Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dry Eye ◽  
Lacrimal Gland ◽  
Culture Media ◽  
Calcium Influx ◽  
Eye Disease ◽  
Specific Marker ◽  
Gland Tissue ◽  
Lysosomal Protein

Abstract Background: Tear deficiency due to dysfunction of the lacrimal gland (LG) is one of the major causes of dry eye disease (DED). Therefore, LG stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue, but the number of stem cells in the LG tissue is pretty low, and 2D long-term cultivation results in the reduction of the differentiation capacity of stem cells. Whereas, 3D LG organoids could be an alternative for a DED therapeutic method, because it is capable of prolonged growth while maintaining the characteristics of the LG tissue. In this study, we developed LG organoids and applied them as cell therapeutics. Methods: Digested cells from human LG tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, expression of specific markers and histological characters were analyzed to prove the LG organoids. And then, the secretory function of LG organoids was confirmed by calcium influx or proteomics after pilocarpine treatment. To explore the curability of developed organoids, finally, mouse-derived lacrimal gland organoids were fabricated and transplanted into mouse lacrimal tissue with DED. Results: The histological features and specific marker expression of LG organoids were similar to normal LG tissue. In the pilocarpine treated LG organoid, internal Ca2+ ions and β -hexosaminidase as known a lysosomal protein in tear fluid was increased. Also, the secreted proteins from pilocarpine treated lacrimal organoid were identified through proteomics. More than 70% of the identified proteins were proven to exosome through GO analysis. These results indicate that our developed organoid has a reactivity to pilocarpine, which shows the function of the lacrimal gland. Additionally, we developed LG organoids from Sjogren’s syndrome patients (SS) and confirmed that their histological feutures similar to that of SS-derived LG tissue. Finally, we confirmed that organoids were well engrafted in the lacrimal tissue at 2 weeks after transplantation of mouse LG orgnoid. Conclusion: This current study demonstrate that our established lacrimal gland organoids resemble characteristics of normal lacrimal gland tissue and could be used as cell therapy for patients with dry eye syndrome.

scholarly journals Establishment of functional epithelial organoids from human lacrimal glands

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sang Yun Jeong ◽  
Woo Hee Choi ◽  
Seong Gyeong Jeon ◽  
Sookon Lee ◽  
Jong-Moon Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture Media ◽  
Calcium Influx ◽  
Specific Marker ◽  
Histological Features ◽  
Hexosaminidase A ◽  
Histological Characteristics ◽  
Marker Expression ◽  
Lysosomal Protein

AbstractBackgroundTear deficiency due to lacrimal gland (LG) dysfunction is one of the major causes of dry eye disease (DED). Therefore, LG stem cell-based therapies have been extensively reported to regenerate injured lacrimal tissue; however, the number of stem cells in the LG tissue is low, and 2D long-term cultivation reduces the differentiation capacity of stem cells. Nevertheless, 3D LG organoids could be an alternative for a DED therapy because it is capable of prolonged growth while maintaining the characteristics of the LG tissue. Here, we report the development of LG organoids and their application as cell therapeutics.MethodsDigested cells from human LG tissue were mixed with Matrigel and cultured in five different media modified from human prostate/salivary organoid culture media. After organoid formation, the growth, specific marker expression, and histological characteristics were analyzed to authenticate the formation of LG organoids. The secretory function of LG organoids was confirmed  through calcium influx or proteomics analysis after pilocarpine treatment. To explore the curability of the developed organoids, mouse-derived LG organoids were fabricated and transplanted into the lacrimal tissue of a mouse model of DED.ResultsThe histological features and specific marker expression of LG organoids were similar to those of normal LG tissue. In the pilocarpine-treated LG organoid, levels of internal Ca2+ions and β-hexosaminidase, a lysosomal protein in tear fluid, were increased. In addition, the secreted proteins from pilocarpine-treated lacrimal organoids were identified through proteomics. More than 70% of the identified proteins were proven to exosome through gene ontology analysis. These results indicate that our developed organoid was pilocarpine reactive, demonstrating the function of LG. Additionally, we developed LG organoids from patients with Sjogren’s syndrome patients (SS) and confirmed that their histological features were similar to those of SS-derived LG tissue. Finally, we confirmed that the mouse LG organoids were well engrafted in the lacrimal tissue two weeks after transplantation.ConclusionThis study demonstrates that the established LG organoids resemble the characteristics of normal LG tissue and may be used as a therapy for patients with DED.

The size of extracellular vesicles secreted by different types of stem cells

2018 ◽  
pp. 38-42
Author(s):  
И.Б. Алчинова ◽  
М.В. Полякова ◽  
И.Н. Сабурина ◽  
М.Ю. Карганов
Keyword(s):  
Stem Cells ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Living Organism ◽  
Isolation And Characterization ◽  
Transmission Electron ◽  
Study Results ◽  
Multipotent Mesenchymal Stem Cells ◽  
Rat Adipose Tissue ◽  
Almost All

Механизм терапевтического действия мультипотентных мезенхимных стволовых клеток (ММСК) на облученный организм в последнее время вызывает повышенный интерес исследователей. В качестве активного участника паракринного механизма реализации этого эффекта предлагают рассматривать внеклеточные везикулы, секретируемые практически всеми клетками живого организма. Цель работы: выделить и охарактеризовать внеклеточные везикулы, продуцируемые стволовыми клетками различной природы. Материалы и методы. Суспензии внеклеточных везикул, выделенных по модифицированному протоколу дифференциального центрифугирования из культуральных жидкостей от культур ММСК костного мозга человека 2-го пассажа и ММСК жировой ткани крысы 4-го пассажа, были проанализированы методом просвечивающей электронной микроскопии и методом анализа траекторий наночастиц. Результаты. Исследование показало наличие в обоих образцах микрочастиц размерами до и около 100 нм, однако процентное содержание частиц разных размеров в суспензии различалось для двух анализируемых типов клеток. Заключение. Полученные результаты могут свидетельствовать о специфике секреции, обусловленной клеточным типом. A mechanism of the therapeutic effect of multipotent mesenchymal stem cells (MMSC) on irradiated body has recently arisen much interest of researchers. Extracellular vesicles (EVs) secreted by almost all cells of a living organism were suggested to actively contribute to the paracrine mechanism of this effect. The aim of the study was isolation and characterization of extracellular vesicles produced by various types of stem cells. Materials and methods. Suspensions of EVs were isolated from culture media of passage 2 human bone marrow-derived MMSC and passage 4 rat adipose tissue-derived MMSC using a modified protocol of differential centrifugation and then studied using transmission electron microscopy and nanoparticle tracking analysis. Results. The study showed the presence of microparticles with a size of >100 nm in the examined samples. However, the percent content of particles with different sizes in the suspension was different in two analyzed types of cell culture. Conclusion. The study results might reflect a specificity of secretion determined by the cell type.

Isolation, Characterization, and Differentiation of Stem Cells From Various Dental Sources: An In Vitro Study

2021 ◽  
pp. 232020682110107
Author(s):  
Sandeep S. Katti ◽  
Kishore Bhat ◽  
Chetana Bogar
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Statistical Analysis ◽  
Specific Growth ◽  
Culture Media ◽  
In Vitro Study ◽  
Central Research ◽  
Cell Lineages ◽  
Apical Papilla

Aim: The aim of the current study was to isolate stem cells from various dental sources such as dental pulp, periodontal ligament (PDL), and apical papilla, and to characterize stem cells by staining for the presence/absence of specific surface markers and also to differentiate stem cells into osteogenic, chondrogenic, and adipogenic cell lineages by exposing them to specific growth factors under the ideal conditions. Materials and Methods: A total of 117 samples were included in the study, consisting of 30 pulp, 50 gingival, 35 PDL, and 2 apical papilla samples. The pulp was extirpated and transported to the Central Research Laboratory. Gingival connective tissue was collected from the participants undergoing any crown lengthening procedure or any gingivectomy procedure from the Department of Periodontology. A similar procedure was also followed for apical papilla and PDL. Isolation was done followed by the identification of the cells by immunocytochemistry using different markers. Once the identity of cells was confirmed, these cells were treated with different culture media to attain 70% to 100% confluency. Then the medium was replaced with a conditioning medium containing specific growth factors for differentiation into osteogenic, chondrogenic, and adipogenic cell lineages. Result: In our study, the number of samples collected and processed was 117. The isolation rate of stem cells from the above-collected samples was 70%. Statistical analysis—no statistical analysis was done as there was no variability expected. Conclusion: Our study showed that stem cells could be isolated, differentiated, and characterized from different dental sources.

scholarly journals T9. EPIGENETIC PROFILING IN SCHIZOPHRENIA DERIVED HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS) AND NEURONS

2020 ◽  
Vol 46 (Supplement_1) ◽  
pp. S234-S234
Author(s):  
Lorna Farrelly ◽  
Shuangping Zhang ◽  
Erin Flaherty ◽  
Aaron Topol ◽  
Nadine Schrode ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Neural Progenitor Cells ◽  
Gene Expression Pattern ◽  
Early Gene ◽  
Label Free ◽  
Induced Pluripotent

Abstract Background Schizophrenia (SCZ) is a severe psychiatric disorder affecting ~1% of the world’s population. It is largely heritable with genetic risk reflected by a combination of common variants of small effect and highly penetrant rare mutations. Chromatin modifications are known to play critical roles in the mediation of many neurodevelopmental processes, and, when disturbed, may also contribute to the precipitation of psychiatric disorders, such as SCZ. While a handful of candidate-based studies have measured changes in promoter-bound histone modifications, few mechanistic studies have been carried out to explore how these modifications may affect chromatin to precipitate behavioral phenotypes associated with the disease. Methods We applied an unbiased proteomics approach to evaluate the epigenetic landscape of SCZ in human induced pluripotent stem cells (hiPSC), neural progenitor cells (NPCs) and neurons from SCZ patients vs. matched controls. We utilized proteomics-based, label free liquid chromatography mass spectrometry (LC-MS/MS) on purified histones from these cells and confirmed our results by western blotting in postmortem SCZ cortical brain tissues. Furthermore we validated our findings with the application of histone interaction assays and structural and biophysical assessments to identify and confirm novel chromatin ‘readers’. To relate our findings to a SCZ phenotype we used a SCZ rodent model of prepulse inhibition (PPI) to perform pharmacological manipulations and behavioral assessments. Results Using label free mass spectrometry we performed PTM screening of hiPSCs, NPCs and matured neurons derived from SCZ patients and matched controls. We identified, amongst others, altered patterns of hyperacetylation in SCZ neurons. Additionally we identified enhanced binding of particular acetylation ‘reader’ proteins. Pharmacological inhibition of such proteins in an animal model of amphetamine sensitization ameliorated PPI deficits further validating this epigenetic signature in SCZ. Discussion Recent evidence indicates that relevance and patterns of acetylation in epigenetics advances beyond its role in transcription and small molecule inhibitors of these aberrant interactions hold promise as useful therapeutics. This study identifies a role for modulating gene expression changes associated with a SCZ epigenetic signature and warrants further investigation in terms of how this early gene expression pattern perhaps determines susceptibility or severity of the SCZ disease trajectory.

scholarly journals Refractory Dry Eye Syndrome after Transconjunctival Excision of the Palpebral Lobe of the Lacrimal Gland

Medicina ◽  
2021 ◽  
Vol 57 (6) ◽  
pp. 608
Author(s):  
Yong-Jae Lee ◽  
Han-Sol Choi ◽  
Seong-Jun Park ◽  
Hae-Jung Sun ◽  
Sun-Young Jang
Keyword(s):  
Female Patient ◽  
Dry Eye ◽  
Lacrimal Gland ◽  
Medical Center ◽  
Chief Complaint ◽  
Incisional Biopsy ◽  
Lacrimal Glands ◽  
Palpable Mass ◽  
Tear Secretion ◽  
Palpebral Lobe

The aim of the present study was to report two cases of refractory dry eye syndrome (DES) after transconjunctival excision of the palpebral lobe of the lacrimal gland. A 25-year-old female patient with a chief complaint of a palpable mass in both upper eyelids visited our medical center. Preoperative orbital computer tomography showed high-attenuation lesions in both lacrimal glands. Incisional biopsy of the lacrimal gland palpebral lobe via transconjunctival incision was performed in January 2019. At 1 month after the biopsy, a lack of tears and persistent corneal erosions were found in both eyes. Artificial tears, punctal occlusion, autologous serum eye drops, and therapeutic contact lenses were applied in an attempt to control the dry eye symptoms. The patient continues to suffer from intractable DES at 2.5 years after the procedure. The second case involved a 52-year-old female patient who visited our medical center with a chief complaint of a palpable mass in both upper eyelids. Bilateral orbital tumors were diagnosed with preoperative magnetic resonance imaging. An incisional biopsy of the lacrimal gland was performed. Immunoglobulin G4-related dacryoadenitis was confirmed through lacrimal palpebral lobe incisional biopsy. Intractable DES and corneal erosion of her left eye persisted thereafter. A transconjunctival incision is an effective approach for minimizing postoperative scars and is suitable for the biopsy of tumors that are visible through the conjunctiva. After a biopsy of the palpebral lobe of the main lacrimal glands, the secretion of reflex tears decreases due to damage to the secreting ducts of the main lacrimal glands. However, total tear secretion can be maintained by basal tear secretion from the accessory lacrimal glands. In this report, we describe two cases of refractory DES due to decreased total tear secretion, although only the palpebral lobes of the main lacrimal glands were biopsied.

The Influence of Oxygen on the Proliferative Capacity and Differentiation Potential of Lacrimal Gland–Derived Mesenchymal Stem Cells

2015 ◽  
Vol 56 (8) ◽  
pp. 4741 ◽  
Author(s):  
Mathias Roth ◽  
Kristina Spaniol ◽  
Claus Kordes ◽  
Silke Schwarz ◽  
Sonja Mertsch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Lacrimal Gland ◽  
Proliferative Capacity ◽  
Differentiation Potential

scholarly journals Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

2021 ◽  
Vol 32 (1) ◽  
Author(s):  
Mariane Beatriz Sordi ◽  
Raissa Borges Curtarelli ◽  
Izabella Thaís da Silva ◽  
Gislaine Fongaro ◽  
Cesar Augusto Magalhães Benfatti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Culture Media ◽  
Deciduous Teeth ◽  
Free Calcium ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Media Supplementation

AbstractIn in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

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